An important role for CD4 + T cells in adaptive immunity to Toxoplasma gondii in mice lacking the transcription factor Batf3

Immunity t

Frequency of homologous recombination deficiency in a large community-based cohort of epithelial ovarian cancer cases

Purpose/Background: Homologous recombination deficiency (HRD) is a useful predictor of treatment response in patients with epithelial ovarian cancer (EOC). Reported data on the frequency of HRD in EOC is largely based on analysis of patients treated at academic medical centers or who participated in clinical trials. We sought to characterize the frequency of HRD based on mutations in homologous recombination repair (HRR) genes, genomic instability (GI) and loss of heterozygosity (LOH) scores in a large community-based cohort of EOC patients who received genomic testing in the context of routine clinical care. Methods: Information including patient demographic, tumor stage and histology data, and results from ovarian cancer tumor tissue sequencing tests was obtained from the diverse dataset within the Providence St. Joseph Health (PSJH) Electronic Medical Record (EMR) and the system-wide cancer registry data. Patients with an EOC diagnosis (ICD C56.x) during the time interval between January 2015 and January 2020 were included. Structured genomic data was sourced from laboratory information systems and manual abstraction of molecular sequencing reports. Alterations in the following HRR genes were analyzed, along with LOH and GI scores: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L. Results: Within this EOC cohort of 3007 patients, 510 (17%) had tumor tissue sequencing (TTS) ordered in the context of clinical care. TTS increased over time (p\u3c0.0001) and was more frequent in patients with advanced stage disease (33/392 vs 149/659 for Stage I-II vs Stage III-IV, X2=34.6, p \u3c 0.00001) or who had a tissue biopsy at a PSJH facility (214/1,151 vs 139/998 for biopsy vs no biopsy, X2=8.5, p=0.004). Eight patients (1.5%) had an insufficient tissue sample despite multiple attempts. Pathogenic mutations in HRR genes were identified in 97 (19%) patients including BRCA1 (n=54), BRCA 2 (n=25), ATM (n=9), CHEK2 (n=6), PALB2 (n=2) and BRIP1 (n=1). LOH and GI scores reflective of HRD were noted in 34/115 (30%) and 9/40 (23%) of patients tested, respectively. HRR gene mutations and/or GI/LOH were identified in tumors of all stages. Treatment data was available for a subset of patients. Patients with mutations or GI/LOH were far more likely to receive PARPi maintenance therapy than patients without these findings. Conclusion: In this large, community based cohort of EOC cases, commercial TTS identified evidence of HRD in 151 of 510 (27%) patients tested. Molecular alterations were identified in tumors of all stages, suggesting that broad based TTS may be of value. Significance: A large fraction of patients with HRD may not be receiving indicated PARPi therapy. This data suggests there is a need to establish a systemized approach to genetic testing for EOC within the PSJH system. Presenting Author: Nicole Kretzer, MD, PhD, Obstetrics and Gynecology, First Hill Campus, [email protected]

Patterns of genomic testing for epithelial ovarian cancer across a large community-based health care network- a real world experience

Background: NCCN guidelines recommend germline and somatic tumor testing for all women with invasive epithelial ovarian cancer (EOC). Despite this recommendation, testing rates remain low and an optimal strategy to achieve the recommended testing has not been defined. Purpose: We elected to review the patterns of germline and somatic tumor testing for patients with EOC across a large health care network to identify barriers to testing. Methods: Clinical, pathologic, demographic and genomic testing (GT) information including involvement of a genetic counselor, specific test(s) ordered, test vendor, test turn-around time, and test results were obtained from the diverse dataset within the Providence St. Joseph Health (PSJH) Electronic Medical Records and the system-wide cancer registry data mart. PSJH is the third largest non-profit health care system in the US and treats roughly 43,000 cancer patients annually across a 7 state region. Patients with a diagnosis of EOC (ICD C56.x) who had at least a single in-person visit to a PSJH oncology department or a PSJH oncologist for EOC during the period between January 2015 and January 2020 were identified. GT data was manually abstracted, where structured data was un-available; data were analyzed in aggregate and to evaluate for trends over time in patterns of testing. Results: Within this EOC cohort (n=3,007), 1,1027 (34%) had GT results available in the EMR.  Germline testing (GMT) was the initial testing approach in 728 (71%) of women tested, and 210 (29%) of the 728 women who had GMT first went on to have tumor tissue testing (TTT). Of the 300 patients who had TTT first, 79 (26%) went on to have GMT. A BRCA1/2 mutation was identified in 153 (14.9%) patients on GMT and/or TTT. Mutation results were discordant in 7 of 289 (2.4%) who had both GMT and TTT. GT rates increased over time but remained low (46% in 2019). Involvement of a genetic counselor (GC) increased uptake of GT, however, only 62% of patients completed recommended GC referral. GT was ordered from 17 different vendors (12 GMT; 11 TTT). Median time from initial diagnosis to GT order date decreased over time and was 8 weeks and 12 weeks in 2019 for GMT and TTT, respectively. The median time interval between GMT and TTT in patients who had both tests decreased from 130 weeks in 2015 to 6 weeks in 2019. Despite improvement in median time to testing, multiple outliers were observed. Conclusion: The uptake of GT for EOC patients has increased over time but remains low. There is substantial heterogeneity in testing approach including the timing, sequencing, and ordering of tests. Genetic counseling for patients with EOC increases uptake of testing. Significance: These findings highlight the challenges of developing a standardized testing approach across a diverse health care system. There is a need to develop a comprehensive network wide testing strategy that can be effective in multiple settings

Factors impacting genomic testing rates among epithelial ovarian cancer patients across a large community-based healthcare system

Background: Epithelial ovarian cancer (EOC) accounts for the highest mortality of all gynecological cancers. NCCN guidelines recommend germline and somatic testing for all women with invasive EOC. Despite this recommendation, there is a large diversity in the types of testing patients receive even within a single healthcare system. Reported data of genetic testing for epithelial ovarian cancer (EOC) patients is largely based on patients treated at academic medical centers or patients who participate in clinical trials. Purpose: This study sought to determine the rates of germline and somatic testing for epithelial ovarian cancer patients and identify factors that impact testing rates across a large community-based healthcare system over 5 states: WA, OR, CA, AK, and MT. The system is comprised of over 100,000 caregivers, 51 hospitals and 829 physician clinics. The aim was to identify barriers to testing such as region, hospital type, insurance status, racial/ethnic disparities, and stage of diagnosis. Methods: Clinical, pathologic, demographic and genomic testing information was obtained from the diverse dataset within the Providence St. Joseph Health Electronic Medical Records and the system-wide cancer registry for all patients with an EOC diagnosis (ICD C56.x) between January 2015 and January 2020. Structured genomic data was sourced from laboratory information systems and manual abstraction of molecular sequencing reports. This dataset encompasses patient population data among diverse hospital settings and urban/rural environments. Institution types were broken down into academic setting which contain a residency program (Academic), Commission on Cancer (CoC) accredited programs, or smaller community sites (Community) without CoC accreditation. Descriptive statistics and logical regression are utilized to summarize key findings. Results: Within this EOC cohort (3,007 patients), 34% (n=1,027 patients) completed some type of genomic testing (GT). The percentage of patients tested increased from 31% in 2015 to 46% in 2019, reflecting uptake of testing guidelines. The increase in GT rates was largely attributable to an increase in somatic tumor testing (14-39%); while germline testing rates were stable across the interval (25-33%). Patients were more likely to receive testing if they received care at an academic or CoC institution vs community institution (p=0.0001). Logistic regression analysis demonstrated the following factors impacted tested rates: institution type, insurance, and stage at diagnosis (p=0.001, p= 0.0019 and p \u3c 0.0001, respectively). Race/ethnicity did not contribute significantly to the model but did have a significant effect when analyzed independently. Conclusion: This study is the first to analyze practice patterns in GT for EOC across a broad community-based healthcare system servicing 5 states. The data highlight discrepancies in GT heavily influenced by practice setting, insurance status, and stage of diagnosis (likely reflecting payer coverage/ increased need for information in advanced stage disease). Significance: There is a need for a universally defined approach to testing to provide equitable access to evidence based cancer care. Presenting Author: Nicole Kretzer, MD, PhD, Obstetrics and Gynecology, First Hill Campus, [email protected]

Heme-Mediated SPI-C Induction Promotes Monocyte Differentiation into Iron-Recycling Macrophages

Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80^+VCAM1^+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis

Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages

Distinguishing dendritic cells (DCs) from other cells of the mononuclear phagocyte system is complicated by the shared expression of cell surface markers such as CD11c. In this study, we identified Zbtb46 (BTBD4) as a transcription factor selectively expressed by classical DCs (cDCs) and their committed progenitors but not by plasmacytoid DCs (pDCs), monocytes, macrophages, or other lymphoid or myeloid lineages. Using homologous recombination, we replaced the first coding exon of Zbtb46 with GFP to inactivate the locus while allowing detection of Zbtb46 expression. GFP expression in Zbtb46(gfp/+) mice recapitulated the cDC-specific expression of the native locus, being restricted to cDC precursors (pre-cDCs) and lymphoid organ- and tissue-resident cDCs. GFP(+) pre-cDCs had restricted developmental potential, generating cDCs but not pDCs, monocytes, or macrophages. Outside the immune system, Zbtb46 was expressed in committed erythroid progenitors and endothelial cell populations. Zbtb46 overexpression in bone marrow progenitor cells inhibited granulocyte potential and promoted cDC development, and although cDCs developed in Zbtb46(gfp/gfp) (Zbtb46 deficient) mice, they maintained expression of granulocyte colony-stimulating factor and leukemia inhibitory factor receptors, which are normally down-regulated in cDCs. Thus, Zbtb46 may help enforce cDC identity by restricting responsiveness to non-DC growth factors and may serve as a useful marker to identify rare cDC progenitors and distinguish between cDCs and other mononuclear phagocyte lineages

Transcription factor Zeb2 regulates commitment to plasmacytoid dendritic cell and monocyte fate

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow–resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box–binding protein associated with epithelial–mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6C(hi) monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals

Distinct Transcriptional Programs Control Cross-Priming in Classical and Monocyte-Derived Dendritic Cells

Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8+ T cells in response to cell-associated antigens. We found that Ly-6ChiTREML4− monocytes can differentiate into Zbtb46+ Mo-DCs in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) but that Ly-6ChiTREML4+ monocytes were committed to differentiate into Ly-6CloTREML4+ monocytes. Differentiation of Zbtb46+ Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4 and was accompanied by the induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46+ Mo-DCs capable of cross-priming CD8+ T cells. Instead, Irf4−/− monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8+ T cells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs

Transcriptional Control of Dendritic Cell Development

The dendritic cells (DCs) of the immune system function in innate and adaptive responses by directing activity of various effector cells rather than serving as effectors themselves. DCs and closely related myeloid lineages share expression of many surface receptors, presenting a challenge in distinguishing their unique in vivo functions. Recent work has taken advantage of unique transcriptional programs to identify and manipulate murine DCs in vivo. This work has assigned several nonredundant in vivo functions to distinct DC lineages, consisting of plasmacytoid DCs and several subsets of classical DCs that promote different immune effector modules in response to pathogens. In parallel, a correspondence between human and murine DC subsets has emerged, underlying structural similarities for the DC lineages between these species. Recent work has begun to unravel the transcriptional circuitry that controls the development and diversification of DCs from common progenitors in the bone marrow

Opposing Roles of Dendritic Cell Subsets in Experimental GN

Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46-GFP and Snx22-GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c(+) cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46-GFP or Snx22-GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46(+) cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46(+) cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103(+) subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b(+) DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103(+) DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation